Although host T cell immune responses to hepatitis B virus (HBV) have been demonstrated to have important influences on the outcome of HBV infection, the development of T cell epitope-based vaccine and T cell therapy and the clinical evaluation of specific T cell function are currently hampered markedly by the lack of validated HBV T cell epitopes covering broad patients. This study aimed to screen T cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and presenting by thirteen prevalent human leukocyte antigen (HLA)-A allotypes which gather a total gene frequency of around 95% in China and Northeast Asia populations. 187 epitopes were in silico predicted. Of which, 62 epitopes were then functionally validated as real-world HBV T cell epitopes by ex vivo IFN-γ ELISPOT assay and in vitro co-cultures using peripheral blood mononuclear cells (PBMCs) from HBV infected patients. Furthermore, the HLA-A cross-restrictions of each epitope were identified by peptide competitive binding assay using transfected HMy2.CIR cell lines, and by HLA-A/peptide docking as well as molecular dynamic simulation. Finally, a peptide library containing 105 validated epitopes which cross-binding by 13 prevalent HLA-A allotypes were used in ELISPOT assay to enumerate HBV-specific T cells for 116 patients with HBV infection. The spot forming units (SFUs) was significantly correlated with serum HBsAg level as confirmed by multivariate linear regression analysis. This study functionally validated 62 T cell epitopes from HBV main proteins and elucidated their HLA-A restrictions and provided an alternative ELISPOT assay using validated epitope peptides rather than conventional overlapping peptides for the clinical evaluation of HBV-specific T cell responses.
Hepatitis B virus (HBV) infections are still prevalent across the world. The global patients with chronic infection have exceeded 257 million which results in about 880,000 deaths per year due to liver cirrhosis (LC) or hepatocellular carcinoma (HCC). In China, currently about 70 million people have been infected with HBV, around 20-30 million people have suffered from chronic infections which is the main pathogenic factor for 77% of liver cirrhosis and 84% of HCC. Liver inflammation induced by HBV is mainly caused by the host immune responses. Numerous researches have confirmed that HBV-specific T cells not only substantially drive virus clearance and disease progression, but also significantly influence antiviral efficacy and disease recurrence after therapy discontinuation. The immune responses mediated by HBV antigen-specific CD8+ T cells are particularly critical for host antiviral protections since CD8+ cytotoxic T lymphocytes (CTL) are the vital cells to kill virus-infected cells. Human leukocyte antigen (HLA) class I molecules (such as HLA-A, B, and C) expressed by virus-infected liver cells present the HBV epitope peptides to specific CD8+ T cells, thus initiate the activation, proliferation and differentiation of CTLs. However, HLA class I molecules are highly polymorphic in the general population. That means HLA class I allotypes are distinctive from individual to individual, and each HLA allotype presents distinctive epitope peptides, thus leading to different strengths of protective or pathogenic immune responses in different individuals against the same pathogen such as HBV. Thus far, the validated T cell epitopes in HBV antigens are still very limited, only 205 CD8+ T cell epitopes and 79 CD4+ T cell epitopes have been defined from HBV proteome by cellular functional experiments, but most are restricted to several common HLA supertypes, such as HLA-A0201, A2402, B0702, DR04, and DR12 molecules, as displayed in the Hepitopes database and other recent reports. Therefore, the current library of validated HBV T cell epitopes cannot cover a broad population carrying highly polymorphic HLA alleles in the indicated geographical regions, and also can not cover broad antigenic targets recognized by HBV-specific CD8+ or CD4+ T cell clones. This limitation thus hampered the development of T cell epitope-based therapeutic vaccine and T cell immunotherapy, and also hampered the clinical precise evaluation of HBV-specific cellular immunity.
To achieve more variable CD8+ T cell epitopes which cover more dominant HLA-A allotypes and span more HBV proteins, this study dedicated to the mapping of CD8+ T cell epitopes derived from overall HBsAg, HBeAg (covering HBcAg), HBx and HBpol proteins, and restricted by a series of high-frequency HLA-A allotypes which gather a total gene frequency of around 95% in Asian population. 187 epitopes restricted by 13 HLA-A allotypes were in silico predicted and selected as candidate epitopes. Of these, 62 epitopes were then functionally validated as real-world epitopes by ex vivo enzyme-linked immunosorbent spot (ELISPOT) assay and in vitro co-cultures, using patients’ peripheral blood mononuclear cells (PBMCs). Furthermore, the peptide competitive binding assay and HLA-A/peptide docking as well as molecular dynamic simulation were used to identify the binding affinity and HLA-A cross-restrictions of the 62 epitopes. Finally, a peptide library containing 105 validated epitopes was used to enumerate the peripheral HBV-specific T cells in IFN-γ ELISPOT assay for 116 patients with HBV infection.
Patients with HBV infection were recruited in this study from the Division of Hepatitis or Department of Clinical Laboratory at Nanjing Second Hospital. According to the EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection, the CHB patients had clinical, biochemical and virological evidence of chronic hepatitis B infection with HBsAg positiveness for at least 6 months. Three CHB patient groups were enrolled in this study: patients in immune tolerant stage (IT, HBeAg+ chronic infection, high viremia but limited liver inflammation), patients in immune active stage (IA, HBeAg+ chronic hepatitis, high viremia and high level of alanine aminotransferase), and immune inactive carriers (IC or InA, HBeAg- chronic infection, low or undetectable serum viral load and limited liver inflammation with normal level of alanine aminotransferase). In addition, 29 acute resolved patients (R, low or undetectable serum viral load, HBsAg-, and anti-HBcAb+), 20 patients with liver cirrhosis and 14 patients with liver cancer were also enrolled here. The exclusion criteria for these subjects were the infection with hepatitis C virus, hepatitis A virus or human immunodeficiency virus, and malignant tumor.
The present study was conducted according to the Declaration of Helsinki principles, and the human samples collection and use has been approved by Clinical Ethics Committee of Nanjing Second Hospital (ref: 2018-LY-kt054, 2019-LY-ky011, 2021-LS-ky013). For the validation of putative epitopes using IFN-γ ELISPOT assay, 500 whole blood samples from HBV-infected patients were collected from Department of Clinical Laboratory of Nanjing Second Hospital. In this instance, informed consent was waived because these blood samples were the biological specimens obtained from past clinical diagnosis and treatment, but consent was obtained from Clinical Ethics Committee of Nanjing Second Hospital. For the clinical detection of HBV-specific T cells, 116 participants recruited from Division of Hepatitis of Nanjing Second Hospital gave written, informed consent. 3 to 5 mL of whole blood was taken from each patient, and PBMCs were further isolated by density-gradient centrifugation.
T cell epitopes spanning HBsAg, HBeAg (covering HBcAg), HBpol and HBx proteins of HBV and presented by different HLA-A allotypes were in silico predicted using six epitope predication tools and eight types of algorithms (SYFPEITHI, BIMAS, SVMHC-SYFPEITHI/MHCPEP, IEDB-ANN/SMM, NetMHC, EPIJEN). For each HLA-A allotype and each protein, one to five 9-mer and 10-mer peptides with the highest score (highest affinity) as predicted by at least two tools were selected as candidate epitopes for further validation. This study mainly focused on adr and adw serotypes and B, C genotypes of HBV, which are common in Chinese population, and also took A and D genotypes into account. The entire amino acid sequences of each protein from different genotypes were obtained from UniProt database and aligned in Figure S1.
The peptides were synthesized from China Peptides Co., Ltd with a purity of > 95% as defined by HPLC purification and mass spectrometry and were used in cellular functional experiments. Lyophilized peptides were reconstituted at a stock concentration of 2 mg/mL in DMSO-PBS solution and stored in aliquots at -80°C.
Peripheral blood samples from patients with HBV infection were collected from Department of Clinical Laboratory at Nanjing Second Hospital and processed into PBMCs. The predicted epitopes restricted by the indicated HLA-A allotypes were grouped into several peptide pools (10 epitopes/pool, 10 μg/epitope/mL), and co-cultured with PBMCs (2×10 5/well/peptide pool) in 96-well plates coated with anti-human IFN-γ for 20 h followed by IFN-γ ELISPOT assay, according to manufacturer’s instructions (Dakewe Biotech, Shenzhen, China). In parallel, negative control well (PBMCs alone) and positive control well (PBMCs with phytohemagglutinin, PHA, 2.5 μg/mL) were also performed. Notably, in each negative control well, DMSO was supplemented to make its concentration equal to the peptide pool/PBMCs co-culture well. The spot forming units (SFUs) were imaged and enumerated. Positive T cell response was defined according to the criterion as follows: (SFUs in peptide well - SFUs in negative control well) ≧ 5, while SFUs in negative control well was 0-5; or (SFUs in peptide well)/(SFUs in negative control well) ≧ 2, while SFUs in negative control well was > 5. When the peptide pool can significantly stimulate patient’s PBMCs producing IFN-γ, the PBMCs were re-collected from the same individual, then co-cultured with each epitope peptide in the positive peptide pool and followed by IFN-γ ELISPOT assay to identify whether the indicated epitope peptide inducing T cell responses.
Meanwhile, for the patients with positive T cell responses in IFN-γ ELISPOT assay, HLA-A alleles were identified using polymerase chain reaction-sequencing-based typing (PCR-SBT) method, the gold-standard method recommended by the International HLA Work Group. Primers as described were synthesized by Sangon Biotech Co., Ltd (Shanghai) and displayed in Table S1. The DNA from exon 1 to exon 3 of HLA-A was amplified in PCR using primer combination A1/A3 followed by sequencing using primer combination A2F/A2R for exon 2 and A3F/A3R for exon 3. The sequencing data were aligned with the sequences in the HLA database and analyzed using Lasergene software.
Briefly, PBMCs from the patients with HBV infection were prepared, then seeded in 96-well plates (4×10 5 PBMCs/well) and incubated with a single validated epitope peptide (VEP) (20 μg/mL) which presented by the HLA-A allotypes of the indicated patient as in silico predicted, PHA (10 μg/mL) or no peptide in RPMI1640 culture medium with 10% FBS for 7 days at 37°C, 5% CO 2 incubator. Recombinant human IL-2 (20 IU/mL) was added at day 3. On day 7, the corresponding peptide (40 μg/mL) or no peptide (negative control) was added again for another 16-hour co-culture. Then, BFA/Monensin mixture (eBioscience) was added to the cells and co-cultured for another 6 hrs. After that, cells were harvested, washed and blocked with anti-CD16/CD32 (eBioscience) for 20 min, then stained with FITC-labeled anti-human CD3 and APC-labeled anti-human CD8a monoclonal antibodies (mAbs, Biolegend) for 30 min at 4°C. After washing, cells were fixed and permeabilized following the protocol and were further stained with PE-labeled anti-human IFN-γ (BD Bioscience) for 30 min at 4°C followed by flow cytometry analysis. The frequencies of IFN-γ+ cells in CD3+/CD8+ populations were calculated.
The structure of HLA-A used for receptor was obtained in two ways. For HLA molecules with different binding conformations in world Protein Data Bank (PDB, https://www.frankenthalerfoundation.org such as HLA
